A Three-Dimensional Printed Polycaprolactone Scaffold Combined with Co-Axially Electrospun Vancomycin/Ceftazidime/Bone Morphological Protein-2 Sheath-Core Nanofibers for the Repair of Segmental Bone Defects During the Masquelet Procedure.

INTRODUCTION: Masquelet proposed a new solution for the healing of segmental bone defects, thus minimizing the disadvantages associated with traditional bone grafting. However, a major factor leading to the failure of this technique pertains to be the residual infection. Accordingly, we developed an antibiotic- and osteo-inductive agent-loaded composite scaffold to solve this problem. METHODS: A mesh-like polycaprolactone scaffold was prepared using a lab-exploited solution-type three-dimensional printer, and hybrid sheath-core structured poly(lactic-co-glycolic-acid) nanofibers were fabricated using co-axial electrospinning technology. Vancomycin, ceftazidime, and bone morphological protein (BMP)-2 were employed. The in vitro and in vivo (rabbit fracture model) release patterns of applied agents from the composite scaffold were investigated. RESULTS: The results revealed that the drug-eluting composite scaffold enabled the sustainable release of the medications for at least 30 days in vitro. Animal tests demonstrated that a high concentration of medications was maintained. Abundant growth factors were induced within the bioactive membrane stimulated by the applied scaffold. Finally, satisfactory bone healing potential was observed on radiological examination and biomechanical evaluation. DISCUSSION: The developed composite scaffold may facilitate bone healing by inducing bioactive membrane formation and yielding high concentrations of antibiotics and BMP-2 during the Masquelet procedure.

Effects of hyperbaric oxygen on the osteogenic differentiation of mesenchymal stem cells.

BACKGROUND: Hyperbaric oxygenation was shown to increase bone healing in a rabbit model. However, little is known about the regulatory factors and molecular mechanism involved.We hypothesized that the effect of hyperbaric oxygen (HBO) on bone formation is mediated via increases in the osteogenic differentiation of mesenchymal stem cells (MSCs) which are regulated by Wnt signaling. METHODS: The phenotypic characterization of the MSCs was analyzed by flow cytometric analysis. To investigate the effects of HBO on Wnt signaling and osteogenic differentiation of MSCs, mRNA and protein levels of Wnt3a, beta-catenin, GSK-3beta, Runx 2, as well as alkaline phosphatase activity, calcium deposition, and the intensity of von Kossa staining were analyzed after HBO treatment. To investigate the effects of HBO on Wnt processing and secretion, the expression of Wntless and vacuolar ATPases were quantified after HBO treatment. RESULTS: Cells expressed MSC markers such as CD105, CD146, and STRO-1. The mRNA and protein levels of Wnt3a, ß-catenin, and Runx 2 were up-regulated, while GSK-3ß was down-regulated after HBO treatment. Western blot analysis showed an increased ß-catenin translocation with a subsequent stimulation of the expression of target genes after HBO treatment. The above observation was confirmed by small interfering (si)RNA treatment. HBO significantly increased alkaline phosphatase activity, calcium deposition, and the intensity of von Kossa staining of osteogenically differentiated MSCs. We further showed that HBO treatment increased the expression of Wntless, a retromer trafficking protein, and vacuolar ATPases to stimulate Wnt processing and secretion, and the effect was confirmed by siRNA treatment. CONCLUSIONS: HBO treatment increased osteogenic differentiation of MSCs via regulating Wnt processing, secretion, and signaling.

Evaluation of differentially expressed genes by shear stress in human osteoarthritic chondrocytes in vitro.

BACKGROUND: The pathogenesis of osteoarthritis is related to abnormal mechanical stresses that alter cartilage metabolism and chondrocyte survival. Among the mechanical stresses, shear stress is held responsible for the development of arthritis. METHODS: Monolayer cultures of human osteoarthritic chondrocytes were subjected to fluid-induced shear stress in vitro. A cDNA microarray technology was used to screen the differentially regulated genes and quantitative real-time poly-merase chain reaction (Q-RT-PCR) was used to confirm the results. The significance of the expression ratio for each gene was determined on the lowest associated false discovery rate calculated from the changes of gene expression in relation to the standard deviation of repeated measurements for that gene. RESULTS: Exposure of human osteoarthritic chondrocytes to shear stress (0.82 Pa) for 2 hours differentially regulated 373 and 227 clones in two independent microarray analyses with at least a 1.7-fold change. By comparing the differentially regulated clones, 14 upregulated and 6 downregulated genes were identified. Many of the differentially expressed genes were related to cell proliferation/differentiation (TGF-beta, acidic FGF), cell survival/apoptosis (CYP1B1, BCL2L3, TNFRSF11B, chemokine ligands, ADM), and matrix homeostasis (DCN, SDC2, MGP, WISP2). CONCLUSION: The gene expression patterns following shear stress show a high similarity to the gene expression in the reparative process of osteoarthritis chondrocytes. Using microarray analysis, this study suggests a close interaction between shear stress and the pathogenesis of osteoarthritis.